5 The HLA class II component of susceptibility to rheumatoid arthritis B. P A U L W O R D S W O R T H MIKE SALMON

Any consideration of the role of genetic factors in rheumatoid arthritis must take account of the fact that very little is known with certainty about the aetiology of this condition. Consequently, hypotheses about its causes and pathogenesis must remain highly speculative until a fuller picture can be determined. These arguments apply to typical examples of the wellestablished classical forms of the disease; they are perhaps even more pertinent when attempting to distinguish factors which may be relevant to the ultimate prognosis of patients with early synovitis. The particular problems posed by this question relate as much to defining the disease as to any complicated considerations of molecular biology or statistical methodology. In this chapter we will review the evidence that loci within the class II legion of the major histocompatibility complex (MHC) are involved in producing susceptibility to rheumatoid arthritis, and discuss their possible significance as determinants of disease persistence and severity. RHEUMATOID ARTHRITIS: THE CLINICAL SPECTRUM

Rheumatoid arthritis is characterized by a T cell mediated chronic synovitis leading to progressive joint destruction. The considerable clinical and immunopathological heterogeneity within this condition has prompted a number of attempts to refine its definition by the application of strict diagnostic criteria. In particular, the relatively mild forms of synovitis that were categorized as 'possible' or 'probable' rheumatoid arthritis by the 1958 American Rheumatism Association (ARA) criteria (Ropes et al, 1958) can include many other diagnoses, including self-limiting viral arthropathies. A major degree of heterogeneity could present a significant obstacle to the elucidation of relevant aetiological factors, particularly the immunogenetics of rheumatoid arthritis. In this respect the 1987 American College of Rheumatologists (ACR) criteria for rheumatoid arthritis cannot really be regarded as a great advance because they lack specificity, particularly where dealing with mild or early disease. The use of more stringent derivatives of these criteria (Wordsworth et al, 1989) has enabled the definition and study Bailli~re' s Clinical Rheumatology--

Vol. 6, No. 2, June 1992 ISBN 0-7020-1636-5

325 Copyright9 1992,by Bailli6reTindall All rights of reproductionin any formreserved

326

B.P. WORDSWORTHAND M. SALMON

of more homogeneous populations of patients and facilitated the identification of some relevant immunogenetic factors in rheumatoid arthritis.

GENETICS OF RHEUMATOID ARTHRITIS Familial aggregation of rheumatoid arthritis is largely restricted to the families of probands with relatively severe disease (erosive and seropositive), suggesting that the genetic component of the disease is most relevant to disease severity or chronicity (Lawrence, 1970). Indeed, the recurrence risk in siblings of affected probands with mild disease is barely greater than that of the general population. The observation that no more than 30% of the identical co-twins of probands with rheumatoid arthritis also develop the disease is a clear indication of the importance of non-genetic factors. However, since concordance rates for rheumatoid arthritis are approximately five times higher in identical twins of probands than in non-identical twins, it can be inferred that genetic factors are indeed significant (Lawrence, 1970; A h o et al, 1986). In the last two decades, most of the interest in the genetic component to susceptibility has centred on genes within the M H C towards the centromeric end of the short arm of chromosome 6. This region is known to contain many loci important in determining host immune responses (Figure 1). In 1973,

DP

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RING

A2 A1 B2 B1

I

DQ

I

DRB

[

A2 B2 B3

1210

11~] B1A1 B1 B4

DRA

500

1000 HSP-70

I C4B C4A 1000

HLA-B HLA-C

2000

Bf C2

I 2 1

TNF

I

I AB

1500

HLA-X HLA-E

3000

2000 HLA-H HLA-F HLA-A HLA-G

4000

Figure 1. A map of genes present within the human MHC. HSP = heat shock protein; TNF = tumour necrosis factor. After Trowsdale et al (1991).

H L A CLASS n A N D R H E U M A T O I D ARTHRITIS

327

the study of immune response genes in the rheumatic diseases was given a considerable impetus by the discovery of the strong association between ankylosing spondylitis and the human leucocyte antigen (HLA) class I antigen B27, which confers a relative risk of approximately 120 for this disease. Subsequently, considerable effort was made to determine whether similar associations between rheumatoid arthritis and genes within the MHC could be identified. However, associations with H L A class I antigens were uniformly weak or lacking. The first evidence that class II antigens might be relevant in this disease came with the description by Stastny (1974) that lymphocytes from patients with rheumatoid arthritis were frequently mutually non-stimulatory in mixed lymphocyte culture. Population and family studies have subsequently demonstrated conclusively that at least one of the genetic elements concerned in rheumatoid arthritis maps to the MHC (Tiwari and Terasaki, 1985; Jaraquemada et al, 1986; Payami et al, 1986; Wordsworth and Bell, 1992). The MHC spans 3.3 megabases of DNA. It contains three main regions (class I, class II and class III), each of which includes a series of homologous loci (Figure 1). In addition to the well-known H L A class I and class II genes, there are many other loci potentially involved in immune and inflammatory responses. These include components of the classical (C2, C4) and alternative (properdin, factor B) complement pathways, tumour necrosis factor (TNF) (Spies et al, 1986), HSP 70 (a member of the highly conserved heat shock protein family) (Sargent et al, 1989b) and several recently described genes which may be involved in antigen processing and transport (Robertson, 1991). The class I H L A loci (HLA A, H L A B and H L A C) encode transmembrane glycoproteins of the immunoglobulin superfamily which bind and present endogenously synthesized antigens, including those from virally infected and tumour cells, to cytotoxic T cells which express the CD8 molecule. The polymorphic heavy chains of class I molecules associate with non-polymorphic [32-microglobulin prior to expression on the cell surface. Class II molecules, in contrast, are a/j3 heterodimers with their polymorphism concentrated in, but not exclusive to, the [3 chains. Class II MHC molecules present largely exogenous antigens to CD4-bearing helper T cells. In recent years much has been learned of the structure and functional interactions of the H L A class I molecules with both antigens and T cells. With the elucidation of the crystallographic structure of some class I molecules (HLA A2, H L A Aw68 and H L A B27) it has been possible to infer that the membrane-distal portion of the heavy chain is specialized for the binding of small peptide antigens probably eight or nine amino acids in length (Bjorkman et al, 1987; Jardetsky et al, 1991). The antigen binding site is a surface cleft in the molecule bounded on either side by antiparallel o~ helices and in the floor by eight [3pleated sheets (Figure 2). Polymorphism in 9 the H L A class I heavy chains is almost exclusively restricted to the cx~and or2 domains and, furthermore, to those residues which point towards the antigen binding site or the T cell receptor. They are therefore likely to influence peptide binding and/or interactions with T cell antigen receptors. By

328

B. P. W O R D S W O R T H A N D M. S A L M O N

analogy much can be inferred of the likely structure of the H L A class II molecule (Brown et al, 1988) (Figure 2). Although it is now realized that class I molecules play a central role in the presentation of peptide antigens, they were initially recognized as transplantation antigens responsible for triggering the rejection of donor tissue. Their polymorphism has been defined mainly by serological reagents, which form the mainstay of tissue matching prior to organ transplantation. When similar reagents became available for H L A class II typing it was apparent that the major association in rheumatoid arthritis was with H L A class II, and in particular with the DR4 antigen (Stastny, 1974) . . . . . ", so

Y'"

9

,

9

N~

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70

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.

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2. T h e t h r e e - d i m e n s i o n a l s t r u c t u r e o f a class II H L A m o l e c u l e . T h e t h i r d allelic hypervariable region of molecules associated with rheumatoid arthritis have a conserved s e q u e n c e b e t w e e n p o s i t i o n s 66 a n d 75 o f t h e D R I 3 c h a i n . S i g n i f i c a n t n o n - c o n s e r v a t i v e substit u t i o n s a r e u n d e r l i n e d . It is p a r t i c u l a r l y i n t e r e s t i n g t h a t r h e u m a t o i d a r t h r i t i s - a s s o c i a t e d m o l e c u l e s s u c h as D R 1 , D R 4 / D w 4 a n d D R 6 / D w 1 6 a r e v e r y d i f f e r e n t e l s e w h e r e , p a r t i c u l a r l y in t h e f l o o r o f t h e a n t i g e n b i n d i n g cleft, w h i l e m o l e c u l e s s u c h as D R 4 / D w l 0 a n d D R 4 / D w l 3 differ f r o m o t h e r D R 4 m o l e c u l e s o n l y in this r e g i o n ; t h e s e alleles, h o w e v e r , a r e n o t a s s o c i a t e d w i t h the disease.

H L A C L A S S II A N D R H E U M A T O I D ARTHRITIS

329

THE HLA CLASS II REGION AND SUSCEPTIBILITY TO RHEUMATOID ARTHRITIS The HLA class II region comprises approximately 1 million base pairs at the centromeric end of the MHC and can be conveniently divided into DR, DQ and DP subregions. In addition, a number of other class II loci are present, not all of which are functional. It seems likely that the density of genes in the MHC could be as high as one per 25 kilobases of DNA (Sargent et al, 1989a), and a number of 'novel' genes have recently been identified by cosmid mapping. Pulsed-field gel electrophoresis has demonstrated that some DR haplotypes contain inserts relative to others; DR4 has a l l 0 k b insert between DR and DQ, while DR2 contains a 20 kb insert in the same position (Dunham et al, 1989b). HLA class II molecules, like their class I counterparts, are transmembrahe glycoproteins of the immunoglobulin superfamily which are intimately concerned with the presentation of peptide antigens to T lymphocytes in an HLA-restricted fashion. In this case antigens are presented to CD4-positive helper T cells, and under the appropriate circumstances recognition of this antigen/class II HLA complex by a T lymphocyte can lead to its activation, and consequent initiation of an immune response. Rheumatoid arthritis may represent an aberrant autoimmune reaction initiated in some way by the activation of T cells with specificity for selfantigen(s). Although relatively little is known of the T lymphocytes and nothing of the antigen(s) involved in this response, substantial progress has been made in determining the nature of the HLA restriction element. Comparisons of the amino acid sequences of rheumatoid arthritis-associated and nonassociated subtypes of DR4 reveal important differences around the putative antigen binding site that may be important in influencing susceptibility (Figure 2). The first and second hypervariable regions of the [3chains of all DR4 molecules are identical and have been modelled to lie in the floor of the putative antigen binding cleft. Differences are limited to the third allelic hypervariable region which lies along one side of the binding site in the a helical region between amino acids 57 and 86 (Gregersen et al, 1986). Those DR4 subtypes associated with rheumatoid arthritis show striking conservation of the amino acid sequence in this region (Figure 2). For instance, Dw4 differs from Dwl4 only at position 71 with the conservative substitution of lysine for arginine, both of which are basic amino acids. Dwl5 is identical to Dw14 except at position 57, right at one end of the helical domain. In contrast, Dwl0, which is not associated with rheumatoid arthritis, exhibits two charged amino acid substitutions (position 70 and 71) and also a more conservative change (isoleucine for leucine at position 67). Dw13 also exhibits a charge change at position 74 (alanine to glutamic acid). The side chains of the amino acids at positions 74 and 71 are probably orientated towards the peptide binding site, and these changes could therefore substantially alter the peptide-binding characteristics of these molecules. If rheumatoid arthritis can be triggered by the presentation of an 'arthritogenic peptide antigen' to an immune system

330

B.P.

W O R D S W O R T H A N D M. S A L M O N

primed to react in an autoimmune fashion, changes to the DR4 molecule which alter peptide binding are likely to have profound effects on susceptibility to the disease. Similar arguments can be advanced to explain the associations which have been described between certain other HLA DR alleles and rheumatoid arthritis. Weak associations with HLA DR1 have been shown in several studies in different racial groups (Woodrow et al, 1981; Schiff et al, 1982; Nepom et al, 1989; Wordsworth et al, 1989), although this association tends to be masked in Caucasoids by the heavy preponderance of DR4 alleles in the rheumatoid population (Wordsworth et al, 1991). In a few examples there are also associations with the relatively uncommon DRwl0 specificity (Sanchez et al, 1990; Oilier et al, 1991; Wordsworth et al, 1991). Finally, among the Yakima Amerindians, in whom rheumatoid arthritis is relatively common, an intriguing association with the rare DR6/Dw16 haplotype has been described recently (Willkens et al, 1991). Although DR1, DR6 and DRwl0 all exhibit substantial differences in the floor of the putative binding site, they show considerable homology within the third hypervariable region with other rheumatoid-associated alleles (Dw14, Dw15 and Dw4), as illustrated in Figure 2. By analogy with the binding of peptides to HLA class I molecules (Falk et al, 1991), there may be important peptide motifs which allow them to bind to certain class II molecules but not others. Thus, a conserved binding pocket around position 71 in the class II molecule could, theoretically, be a prerequisite for binding a particular 'arthritogenic peptide' in rheumatoid arthritis. However, there are important differences in the binding of peptides to class I and class II molecules: binding is more promiscuous to the latter and peptides tend to be longer at 13 to 17 amino acids (Rudensky et al, 1991). The HLA DR component of susceptibility to rheumatoid arthritis is contributed in a dominant fashion; those alleles (e.g. DR4/Dwl0 and DR2) which are negatively associated with the disease do not provide dominant protection. This contrasts with the dominant protective effect that has been observed from certain HLA DQ molecules, characterized by the presence of aspartic acid at position 57 of the DQ [3 chain, in insulin-dependent diabetes mellitus (Todd et al, 1987). Clearly, the HLA component of rheumatoid arthritis is but one of a number of factors in this disorder; the HLA-linked contribution is probably no more than 30% of the total genetic effect (Deighton et al, 1989). Almost certainly other loci are involved in this process, but it wouldbe unwise to exclude the possibility that other loci within the MHC could also be involved. There is a good precedent for this in coeliac disease, where a clear association with a particular HLA class II allele (DQw2) exists (Sollid et al, 1989) but, in addition, another locus on the extended B8, DR3, DQw2 haplotype is also implicated (Rosenberg et al, 1989), as illustrated in Table 1. To date there is relatively little evidence in rheumatoid arthritis to support the concept of a second operant locus within the HLA class II region or nearby. Some interest has focused on a possible effect from the DQ locus (DQw7), particularly in relation to disease severity (Sansom et al, 1989;

331

H L A C L A S S II A N D R H E U M A T O I D ARTHRITIS

Table 1. The associationof coelicdisease with the extended haplotype HLA B8, DR3, DQw2. DR3-positivepatients accountfor 90% of all cases, but DQw2 for 95%, pinpointing the latter as the primary susceptibility allele. However, a second locus restricted to this extended haplotype must also be involved. B8, DR3, DQw2

non-B8, DR3, DQw2

29

0

18

5

DR3 coelic patients (n = 29) DR3 healthy controls (n = 23)

Stevens et al, 1989). In more severe variants of the disease, including Felty's syndrome, a stronger association is apparent with DR4 than in populations with relatively mild disease. The frequency of DR4 in Felty's syndrome may be as high as 95% (Sansom et al, 1987; Lanchbury et al, 1991), while in some community-based surveys of rheumatoid arthritis (including many mild cases of the disease) the DR4 association may not be apparent (De Jongh et al, 1984). In Felty's syndrome DQw7 is also increased (Sansom et al, 1987). H L A DR4 is tightly linked to DQ3 (defined serologically), but this specificity can be subdivided using monoclonal antibodies, restriction fragment length polymorphisms or oligonucleotide probing into several subtypes. In Caucasoids the DQ3 on normal DR4 haplotypes splits either to DQw7 (50%) or DQw8 (50%), The observation that the DQw7 form of DQ3 is increased in DR4-positive Felty's syndrome has led to the suggestion that the DQw7 allele contributes to disease severity in some way. The most likely explanation for these findings probably lies in the subsequent observation that Felty's syndrome is itself preferentially associated with the Dw4 sulStype of DR4 (Lanchbury et al, 1991). This contrasts with the approximately equal association with either Dw4 or Dw14 seen in other variants of the disease. D w l 4 is almost invariably linked to DQw8 in Caucasoids, while Dw4 is linked either to DQw7 or DQw8; consequently, since Dw4 is increased in the Felty's population, a relative increase in the frequency of DQw7 is only to be expected (Table 2). However, leading on from this work, it now appears that the class II association may be somewhat more complex than the relatively straightforward dominant susceptibility which was originally envisaged. Severe and early onset rheumatoid arthritis is more commonly associated with DR4 homozygosity than milder variants of the disease (Nepom et al, Table 2. The primary association of Dw4 with susceptibilityto Felty's syndrome. The preferential linkage of Dw4 to DQw7 rather than DQw8 (approximately2:1) accounts for the secondary association of Felty's syndromewith DQw7. Frequency of HLA DR alleles (%) DR4 DR4/Dw4 DR4/Dwl4 DR4/Dwl0 DR4/Dwl3 Felty's syndrome (n = 43) Controls (n = 107)

93

88

26

0

0

32

20

10

1

3

332

B. P. WORDSWORTH AND M. SALMON

1984, 1986; Lanchbury et al, 1991). A rather surprising observation, given that Dw4 is the commonest subtype of DR4 in the general and rheumatoid populations, is that these apparent DR4 homozygotes tend to be Dw4/Dw14 or even Dw4/Dw15 compound heterozygotes, rather than true Dw4/Dw4 homozygotes (Table 3). These results could be explained either in terms of these particular allelic combinations or effects mediated by linked genes. Since Dw4 is a very strong independent risk factor, we will concentrate on the role of the other haplotype. Table 3. The effect of compound heterozygotes of DR4 on susceptibility to rheumatoid

arthritis. A comparison of the subtype frequencies of DR4 in patients with severe rheumatoid arthritis or Felty's syndrome who are DR4 homozygotes shows a significant distortion of genotypes towards combined alleles such as Dw4/Dwl4 and Dw4/Dwl5. Genotype Dw4/Dw4 Dw4/Dw14 Dw14/Dw14 Dw4/Dw15

Observed

Expected

Probability

(o)

(E)

(O/E)

40 63 2 4

46 36 7 < 0.5

0.8 1.7 (P